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  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
  • C12Q1/6844—Nucleic acid amplification reactions
  • C12Q1/686—Polymerase chain reaction [PCR]

Definitions

• the present invention concerns a simplified method in the field of nucleic acid analytics.
• the method according to the invention enables a gel electrophoretic nucleic acid determination to be carried out without gel staining because the dye is added to the sample containing nucleic acids.
• An electrophoretic separation of nucleic acids in gel matrices usually takes place either in agarose gels or in polyacrylamide gels.
• the resolving power is determined by the content of agarose in the gel in the case of agarose gels, hi contrast, the resolving power of polyacrylamide gels is adjusted by the mixing ratio of acrylamide to bisacrylamide.
• the detection of nucleic acids in nucleic acid analytics with the aid of gel electrophoresis is based on the fact that fluorescent DNA binding dyes permanently bind non-covalently to nucleic acids and in their bound form enable nucleic acids to be located in the gel matrix after excitation with light of a suitable wavelength.
• these dyes can also be used to detect PCR amplificates in real time PCR or qPCR since they do not substantially inhibit the PCR reaction (US 6,569,627).
• SybrGreen and SybrGold which can also be used in real time PCR (Gudnason, H., et al., Nucleic Acids Research 35(19) (2007) el 27).
• certain dyes exist for real time PCR applications which are especially suitable for thermal melting curve analyses such as the LightCycler480 RO 27 dye (Roche Applied Science Cat. No.: 04 909 640 001). They are grouped together as HRM (high resolution melting) fluorescent dyes and are characterized in that they can be used in higher concentrations in the PCR without inhibiting the PCR reaction.
• HRM high resolution melting
• Capillary gel electrophoresis is also an important method for analysing PCR products, for analysing fragments from the restriction digestion, for mutation analytics and DNA sequencing due to its speed, the ability to automate and its high resolving power.
• LIF (laser induced fluorescence) detection plays an important role in this connection in order to make it possible to detect and quantify DNA in a highly sensitive manner and in a high dilution, hi addition to the covalent coupling of fluorescent dyes to the DNA (by means of fluorescent labelled primers during DNA sequencing), the use of dsDNA intercalators plays a major role as a non- covalent method which enables DNA to be detected fluorimetrically and simply by means of stable dye-DNA complexes. The same intercalators are used for this as for the detection of DNA in gel electrophoresis (Sang, F., et al., J. Sep. Sci. 29 (2006) 1275-1280).
• the dyes used for electrophoresis are either admixed with the gel preparation before polymerization in the case of ethidium bromide, or the gel is stained after completion of the gel electrophoresis with the aid of an aqueous dye solution containing ethidium bromide or another dye.
• SybrGreen I can be added to the sample containing the nucleic acid before loading the gel, provided the nucleic acid is incubated with the dye for a further 15 minutes before being applied to the gel (Karlsen, F., et al., Journal of Virol. Methods 55 (1995) 153-156, and Jin, X., et al., FASEB J. 10 Al 128 (1996) abstract # 751).
• the staining methods for the gel electrophoretic analysis of nucleic acids known from the prior art thus have the disadvantage that the addition before polymerization only takes place with the mutagenic substance ethidium bromide, staining in a dye solution is time-consuming and laborious and requires large amounts of dye, or the addition of the dye immediately before loading the gel requires a longer incubation period.
• the present invention therefore concerns a method for analysing nucleic acids in a sample comprising the following steps
• a DNA binding dye containing a benzothiazole, a benzothiazolium or a benzoxazolium group to the sample to be analysed carrying out a polymerase chain reaction applying the sample to a gel matrix separating the nucleic acid molecules according to their size by applying a voltage excitation with light of a suitable wavelength for the optical visualization of the nucleic acid / DNA binding dye complexes.
• the method according to the present invention is particularily characterized in that the gel matrix is not pre-stained prior to the separation of the nucleic acid molecules and further in that the gel matrix is not stained after the separation of the nucleic acid molecules.
• the amplification is measured in real time during the PCR with the aid of the DNA binding dye.
• the gel matrix preferably consists either of agarose or of polyacrylamide. If it is polyacrylamide, the gel matrix can also be in a capillary in order to carry out a capillary gel electrophoresis.
• the DNA binding dyes are preferably selected from the groups consisting of RO27, Syto 9, LC Green and LCGreen+.
• RO 27 is particularly preferred in this case.
• the DNA binding dyes are usually added to the sample at a concentration of 1 - 10 ⁇ M and preferably 2 - 4 ⁇ M.
• Another aspect of the present invention concerns a kit, containing
• - reagents for carrying out a polymerase chain reaction a vessel containing a DNA binding dye with a benzothiazole, benzothiazolium or benzoxazolium group and at least one gel matrix consisting of polyacrylamide.
• the DNA binding dye is in turn preferably selected from a group consisting of RO27, SybrGreen I, SybrGreen II, Syto 9, LC Green and LCGreen+.
• Figure 1 Gel electrophoresis with subsequent detection of the amplificate by a Lumilmager without (left) and with (right) restaining.
• Figure 3 Multiplication series of the molecular weight marker with constant concentrations of 3.2 ⁇ M RO 27 on a 20 % polyacrylamide gel (left) and on a 6 % polyacrylamide gel (right).
• Figure 4a Polyacrylamide gel electrophoresis of PCR mixtures containing
• Figure 4b Polyacrylamide gel electrophoresis of PCR mixtures containing
• Figure 4d Polyacrylamide gel electrophoresis of PCR mixtures containing
• the origin of the present invention was the surprising finding that it is possible to add a real time PCR mixture containing a DNA binding dye directly to the PCR and namely at a concentration which is sufficient to allow omission of a subsequent staining of the PCR products for example with ethidium bromide or with SybrGreen I or SybrGreen II after the gel electrophoresis.
• These DNA binding dyes only emit a corresponding fluorescence signal after excitation with light of a suitable wavelength when they have bound to double-stranded nucleic acid.
• the detectability requires a strong, non-covalent binding of the fluorescent dye to the
• DNA binding dyes migrate in an unbound form towards the cathode during gel electrophoresis.
• the charge of the nucleic acids is, however, not sufficient for the nucleic acid / binding dye complex to migrate towards the anode during a gel electrophoresis.
• the present invention concerns a method for analysing nucleic acids in a sample comprising the following steps
• a DNA binding dye containing a benzothiazolium or benzoxazolium group to the sample to be analysed - carrying out a polymerase chain reaction (PCR) applying the sample to a gel matrix separating the nucleic acid molecules according to their size by applying a voltage excitation with light of a suitable wavelength for the optical visualization of the nucleic acid / DNA binding dye complexes.
• PCR polymerase chain reaction
• the method according to the present invention is particularily characterized in that the gel matrix is not pre-stained prior to the separation of the nucleic acid molecules and further in that the gel matrix is not stained after the separation of the nucleic acid molecules.
• the sample to be analysed is usually a mixture of nucleic acids which have been obtained from biological material such as for example cellular lysates. These lysates can be prepared by any standard methods. As a rule the nucleic acids present in the lysates are at least partially purified by methods known from the prior art before a PCR can be carried out by standard methods (US 4,683,202, US 4,683,195, US 4,965,188). The nucleic acids present in the sample can either be DNA or RNA.
• the PCR is carried out in the form of a one-step RT-PCR in which the RNA is firstly reversely transcribed into a single-stranded cDNA with the aid of suitable enzymes and subsequently this cDNA is amplified to form a double-stranded end product (US 5,407,800, US 5,322,770, US 5,310,652).
• the progress of the PCR reaction can be measured in real time during the amplification with the aid of the DNA binding dye (US 6,569,627).
• This requires thermocyclers with optical modules which excite the DNA binding dye contained in the PCR reaction vessel during the PCR reaction and can subsequently detect the fluorescence signal emitted by this dye (e.g. US 6,814,934, US 6,106,777).
• the DNA binding dyes are usually added to the sample at a concentration of 1 - 10 ⁇ M and preferably 2 - 4 ⁇ M. These concentrations do not usually lead to a measurable or significant inhibition of the polymerase catalysed PCR reaction in the case of the dyes that are to be used according to the invention.
• the DNA binding dyes that are used according to the invention are those dyes whose binding to nucleic acids is not exclusively based on the principle of interacalation. This applies in particular to all fluorescent dyes containing a benzothiazole, benzothiazolium or benzoxazolium group.
• Benzothiazolium has the chemical structure:
• Benzoxazolium has an identical chemical structure except that the sulfur atom is replaced by an oxygen atom in the pentacyclic ring.
• Rl is any substituent and is preferably an optionally substituted Ci - C 6 alkyl.
• Al, A2, A3 and A4 are hydrogen or a representative of Al, A2, A3 is a halogen,
• B is either sulfur or oxygen
• D is either an unsubstituted or a substituted Ci - C 6 alkyl.
• - X is either hydrogen or a methoxy group,
• L is either CH 3 or phenyl
• - M is either phenyl or a substituted or unsubstituted Ci - Ci 8 amino-alkyl. Examples of this are:
• the DNA binding dye which can be by far be used particularly well within the scope of the present invention is the LC480 Resolight Dye (Roche Diagnostics Catalogue No: 04 909 640 001). This dye can be used in high concentrations in the PCR and has the following chemical structure:
• DNA binding dyes that are published and disclosed in WO 06/121423 can be used within the scope of the present method according to the invention.
• Two of these dyes such as for example LCGreen and LCGreen+ are commercially available (Idaho Technology, Cat. No: BCHM- AS Y-0003 and BCHM-ASY-0006).
• LCGreen and LCGreen+ are fluorescent dyes from the group of HRM (high resolution melting) fluorescent dyes which can be used for so-called high resolution melting analyses after a real time PCR. They have in common a low toxicity during the PCR amplification so that they can be used in higher concentrations without inhibiting the PCR.
• Another class of dyes which is suitable for use in a method according to the invention are all dyes that are published and disclosed in US 5,658,751. These are asymmetic cyanine dyes containing a benzothiazole or benzothiazolium group. Representatives of these types of dye are SybrGreen I, SybrGreen II, YO-PRO, TO- PRO, BEBO and BOXTO. Several of these dyes are commercially available, also including SybrGreen I, SybrGreenll, TO-PRO-I, YO-PRO-I, BEBO and BOXTO. SybrGreen I has the following chemical structure:
• BEBO An example of a benzothiazole that can be used within the scope of the method according to the invention is BEBO:
• An additional class of dyes which is suitable for use in the method according to the invention are dyes which contain a benzoxazolium group.
• the following commercially available dyes are particularly suitable: SYTO-9 (Invitrogen Cat. No.
• Ethidium bromide has the structural formula:
• the method according to the invention excludes the use of ethidium bromide or similar intercalating dyes.
• the gel matrix can consist of 0.02 to 6 % w/v and preferably 0.5 to 4 % agarose in a TBE buffer system.
• the electrophoretic separation at 10 - 150 V then preferably takes place in a horizontal direction for 10 to 180 minutes.
• the gel matrix can consist of a mixture of 4 % to 25 % w/v and preferably of 6 - 20 % acrylamide / bisacrylamide in a TAE buffer system.
• a separation time of 10 to 30 minutes is preferred.
• the migration properties are determined in this case by the proportion of acrylamide as well as by the degree of cross-linking. This depends on the mixing ratio between acrylamide and bisacrylamide.
• the electrophoretic separation is carried out at 20 to 250 V for 15 to 180 minutes. A separation time of 15 to 45 minutes is preferred.
• the acrylamide matrix is in a capillary for carrying out a capillary gel electrophoresis (Schwartz, H.E., et al., J. Chromatogr. 559 (1991) 267- 283; Guttman, A., Methods in Molecular Biology 52 (1996) 157-169: Capillary Gel Electrophoresis). It is a major advantage to be able to add these fluorescent dyes before the PCR reaction especially for the highly sensitive, highly resolving and rapid analysis of DNA fragments in the combination of PCR / capillary gel electrophoresis with LIF detection.
• the nucleic acids are detected for example directly by means of a UV transilluminator or with the aid of imaging instruments such as the Lumilmager (Roche Applied Science Cat. No. 2c 012 847).
• a UV transilluminator or with the aid of imaging instruments such as the Lumilmager (Roche Applied Science Cat. No. 2c 012 847).
• the Lumilmager Roche Applied Science Cat. No. 2c 012 847.
• Another aspect of the present invention concerns a kit containing:
• one or more reagents for carrying out a polymerase chain reaction a vessel containing a DNA binding dye with a benzothiazole, benzothiazolium or benzoxazolium group - at least one gel matrix consisting of agarose or polyacrylamide.
• the DNA binding dye is in turn preferably selected from a group consisting of RO 27, SybrGreen I, SybrGreen II, Syto 9, LCGreen, LCGreen+, SYTO-13, SYTO- 16, SYTO-60, SYTO-62, SYTO-64 and SYTO-82 as described for the method according to the invention.
• the gel matrix is shrink-wrapped in a foil to protect it against drying out.
• the kit can contain an appropriate gel electrophoresis run buffer. It does not contain any staining dye.
• the reagents for carrying out the polymerase chain reaction are usually a thermostable DNA polymerase such as for example Taq polymerase or a thermostable polymerase with additional reverse transcriptase activity, deoxynucleoside triphosphates, as well as suitable buffers and PCR additives such as for example magnesium chloride.
• the reagents can be present individually in the kit.
• One or more components including the DNA binding dye can also be present in the kit as a mixture in so-called master mixes, hi addition such a kit can contain components for DNA sample preparation.
• Example 1 Blockcycler PCR in the presence of RO 27 and subsequent analysis of the PCR product by gel electrophoresis:
• Fig. 1 shows the visual result without (left side) and with restaining (right side). It can be seen that the presence of RO 27 alone in the PCR mixture is sufficient to detect the amplicon with adequate sensitivity. In contrast the restaining of the gel with SybrGreen I does not increase the sensitivity.
• SybrGreen I, SybrGreen II or RO 27 before application to a gel system according to example 1.
• the result is shown in fig. 2. It can be seen that in particular SybrGreen I and RO 27 are suitable for a sensitive detection. No DNA is detectable when ethidium bromide is added to the length standard before application to the gel.
• Example 2 was repeated with samples containing in each case 3.2 ⁇ M RO 27 but different amounts of length standards(l-fold to 5-fold) on differently cross-linked gels (6 % and 20 %).
• Fig. 3 clearly shows that at the same molecular weight standard concentration (x 5) and at the same RO 27 concentration (3.2 ⁇ M) the detectability of the 50 - 150 bp standards is only slightly better in the case of the 6 % polyacrylamide gels.
• the method according to the invention can be used independently of the degree of cross-linking of the respective gel system.
• LightCycler TM 20 ⁇ l capillaries
• Template plasmid DNA containing 18s RNA gene sequence in different copy numbers ( 10 4 , 10 3 , 10 2 , 10 ' )
• amplification 1 95 °C 10 sec 20°C/min 60 °C 15 sec 20°C/min ⁇ 10 x

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